Application of Synthetic Biology for Increasing Anaerobic Biodiesel Production in Escherichia coli

64 Pages·2016·2.57 MB·English

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Masters Theses Graduate School
8-2013
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Michael Christopher Wierzbicki
[email protected]
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Wierzbicki, Michael Christopher, "Application of Synthetic Biology for Increasing Anaerobic Biodiesel
Production in Escherichia coli. " Master's Thesis, University of Tennessee, 2013.
https://trace.tennessee.edu/utk_gradthes/2475
This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and
Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE:
Tennessee Research and Creative Exchange. For more information, please contact [email protected].
To the Graduate Council:
I am submitting herewith a thesis written by Michael Christopher Wierzbicki entitled "Application
of Synthetic Biology for Increasing Anaerobic Biodiesel Production in Escherichia coli." I have
examined the final electronic copy of this thesis for form and content and recommend that it be
accepted in partial fulfillment of the requirements for the degree of Master of Science, with a
major in Chemical Engineering.
Cong T. Trinh, Major Professor
We have read this thesis and recommend its acceptance:
Eric Boder, Barry Bruce
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
Application of Synthetic Biology for
Increasing Anaerobic Biodiesel Production
in Escherichia coli

A Thesis Presented for the
Master of Science
Degree
The University of Tennessee, Knoxville

Michael Christopher Wierzbicki
August 2013
Copyright © 2013 by Michael Christopher Wierzbicki
All rights reserved.

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Acknowledgements

I would like to thank Dr. Cong Trinh for employing me in his laboratory, giving me
much-needed experience in research and for serving on my committee. Additionally I would like
to specially thank Dr. Narayan Niraula, Donovan Layton, and Akshitha Yarrabothula for
assisting in all aspects of this project. I would also like to thank Dr. Eric Boder and Dr. Barry
Bruce for serving as the committee members of my thesis defense. Acknowledgement also goes
to the Department of Chemical and Biomolecular Engineering for giving me the opportunity to
pursue a master’s degree.

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Abstract
The ever-increasing demand for transportation biofuels requires new and novel
approaches to solve the complexities associated with efficient biofuel production. Ethanol, the
most common biofuel, has physical limitation associated with difficulty of separations and issues
with water contamination and as such is not a long-term transportation fuel solution. (Lou &
Singh, 2010; Wheals, Basso, Alves, & Amorim, 1999) Biodiesel is seen as a possible alternative
to ethanol due to its hydrophobicity and also has comparable energy density and cetane number
to its petroleum derived counterpart. (Kalscheuer, Stölting, & Steinbüchel, 2006) Because of
feedstock limitations, biodiesel produced from vegetable oils is limited by the supply of
vegetable oil crops which creates scaling issues and land usage concerns. (Kalscheuer et al.,
2006) An alternate method for biodiesel (fatty acid ethyl esters, FAEEs) generation was
proposed which would bypass the need for vegetable oils by utilizing the fatty acids and ethanol
made in engineered Escherichia coli. (Kalscheuer et al., 2006) FAEE is not water soluble, so
water contamination in fuel supplies seen with ethanol is not likely to cause damage to fuel
infrastructure and has similar combustion properties to petroleum based diesel. (Lou & Singh,
2010)
The goal of this project is to apply metabolic engineering and synthetic biology principles
to engineer E. coli for efficient anaerobic production of FAEE from fermentable sugars. The
strain utilized for this project builds upon previous work in which elementary mode analysis was
used to design an E. coli strain that minimizes fermentative by-products under anaerobic
conditions. The engineered strain provided an optimized platform to employ plug-and-play
modular principles for fully endogenous FAEE biosynthesis.
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In order to produce the desired esters, two parallel pathways were introduced, the first
produced fatty-acyl-CoA, and the second the alcohol of interest. These two molecules are then
catalyzed by a wax-ester synthase to produce desired biodiesel. Pathway flux engineering
principles were employed to balance the metabolic fluxes of the two pathways that complete for
the common substrate, pyruvate. The results show the dynamic range of module fluxes that can
be achieved by varying promoter strength, operon orientation, and plasmid copy number.

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Contents
CHAPTER 1 INTRODUCTION ................................................................................................. 1

1.1WHY WE ARE INTERESTED IN DEVELOPING ADVANCED BIOFUEL TECHNOLOGIES ................ 1

1.2 HOW BIODIESEL IS CURRENTLY MADE .................................................................................. 2

1.3 BENEFITS AND DRAWBACKS OF FAEE ................................................................................... 3

1.4 BIOLOGICAL CONVERSION ROUTE FOR PRODUCING FATTY ACID ETHYL ESTER .................... 3

1.5 IMPORTANCE OF ANAEROBIC PRODUCTION FOR BIOFUELS .................................................... 5

1.6 METABOLIC ENGINEERING AND THE ROLE OF METABOLIC NETWORK MODELING ................ 6

1.7 PROJECT SCOPE ...................................................................................................................... 7

1.8 PROJECT CONTRIBUTIONS ...................................................................................................... 7

CHAPTER 2 METHODS ............................................................................................................. 8

2.1 STRAINS AND PLASMIDS ......................................................................................................... 8

2.2 STRAIN CHARACTERIZATION ................................................................................................ 10

2.3 ANALYTICAL METHOD ......................................................................................................... 11

2.4 RESULT REPORTING ............................................................................................................. 12

CHAPTER 3 ENGINEERING THE FAEE-PRODUCING PATHWAY FOR ENHANCED
FAEE PRODUCTION IN E. COLI ........................................................................................... 13

3.1 EXPERIMENTAL TEST PATHWAY .......................................................................................... 13

3.2 BASE CASE STUDY ............................................................................................................... 14

3.3 EFFECT OF PROMOTER SWAPPING ON FAEE PRODUCTION ................................................... 15

3.4 EFFECT OF COPY NUMBER AUGMENTATION ........................................................................ 20

3.5 EFFECT OF OPERON ORIENTATION ON FAEE PRODUCTION .................................................. 22

3.6 EFFECT OF ALTERNATE ETHANOL PATHWAY ON FAEE PRODUCTION ................................. 29

3.7 EFFECT OF THE ENGINEERED STRAIN ON FAEE PRODUCTION .............................................. 32

3.8 EFFECT OF UTILIZATION A MIXED STRAIN CULTURE ON FAEE PRODUCTION ..................... 34

3.9 EFFECT OF VARYING PLASMID COPY NUMBER ON CHAIN LENGTH DISTRIBUTION OF FAEE
................................................................................................................................................... 36

3.10 GENERATING BIODIESEL WITH ENHANCED COMBUSTION PROPERTIES .............................. 38

CHAPTER 4 CONCLUSIONS AND FUTURE WORK ........................................................ 47

REFERENCES ............................................................................................................................ 49

VITA ............................................................................................................................................. 53

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LIST OF TABLES

TABLE 1 GENOTYPE OF TCS083 ΔFADE .......................................................................................... 8

TABLE 2 LIST OF PLASMIDS USED IN THIS STUDY.. ........................................................................... 9

TABLE 3 COMPOSITION OF 5X M9 SALT SOLUTION. ....................................................................... 10

TABLE 4 SUMMARY OF CETANE NUMBERS FOR ESTERS OF DIFFERENT FATTY ACID AND ALCOHOL
MOIETIES. ............................................................................................................................... 39

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LIST OF FIGURES

FIGURE 1 A SIMPLIFIED METABOLIC PATHWAY TO CONVERT GLUCOSE INTO FATTY ACYL-COA. ..... 4

FIGURE 2 A SIMPLIFIED METABOLIC PATHWAY TO CONVERT GLUCOSE INTO ETHANOL. ................... 5

FIGURE 3 A SIMPLIFIED PATHWAY FOR THE PRODUCTION OF FATTY ACID ETHYL ESTERS. ............. 13

FIGURE 4 DNA SEQUENCE ALIGNMENT OF VARIANT LAC PROMOTERS. .......................................... 16

FIGURE 5 THE BASELINE ESTER YIELD EXPERIMENT. ...................................................................... 17

FIGURE 6 EFFECT OF PLASMID COPY NUMBER ON FAEE PRODUCTION. .......................................... 21

FIGURE 7 A REPRESENTATION OF THE SINGLE PLASMID SYSTEM ORIENTATION. ............................. 23

FIGURE 8 EFFECTS OF THE SINGLE PLASMID SYSTEM IN COMPARISON TO THE BASELINE DUAL
PLASMID SYSTEMS ON FAEE PRODUCTION. ............................................................................ 24

FIGURE 9 A PROPOSED CONSTRUCT FOR HIGH EXPRESSION OF THE SECOND OPERON BEYOND THE
LIMITATIONS PRESENT IN CURRENT EXPRESSION MOTIFS. ....................................................... 27

FIGURE 10 THE RELATION BETWEEN PROMOTER STRENGTH AND REPRESSION EFFECT IN THE SINGLE
PLASMID MIXED PROMOTER SYSTEM. ...................................................................................... 28

FIGURE 11 A PROPOSED CONSTRUCT TO DEMONSTRATE AN INVERSE GENE EXPRESSION SYSTEM. . 29

FIGURE 12 EFFECT OF AN ALTERNATE ETHANOL PATHWAY ON FAEE PRODUCTION. ..................... 30

FIGURE 13 EFFECT OF STRAIN ENGINEERING IN COMPARISON TO THE WILD TYPE ON FAEE
PRODUCTION. .......................................................................................................................... 33

FIGURE 14 EFFECT OF SINGLE AND MIXED CELL CULTURES ON FAEE PRODUCTION. ..................... 35

FIGURE 15 EFFECT OF THE FATTY-ACYL COA + ATFA PLASMID COPY NUMBER ON FAEE MOIETY
DISTRIBUTION. ........................................................................................................................ 37

FIGURE 16 PLOTTING THE EFFECT OF FATTY ACID AND ALCOHOL CARBON CHAIN LENGTH ON THE
CETANE NUMBER. ................................................................................................................... 40

FIGURE 17 OVERLAID CHROMATOGRAMS OF DIFFERENT ESTERS PRODUCED BY THE DOPING
EXPERIMENT. .......................................................................................................................... 42

FIGURE 18 OVERLAID HPLC CHROMATOGRAMS DEMONSTRATE THE PRODUCTION OF ISOBUTANOL
AND BUTANOL BY EXPRESSING PCT13 AND PBUTANOL RESPECTIVELY IN TCS83 ΔFADE (DE3)
............................................................................................................................................... 43

FIGURE 19 GCMS CHROMATOGRAM DEMONSTRATES THE PRODUCTION OF ENDOGENOUS ISOBUTYL
PALMITATE. ............................................................................................................................ 46

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